Figure 1.

PMA induces antiproliferative PKCα-ERK signaling in intestinal epithelial cells.A, IEC-18 cells were pretreated with vehicle (−) or 1 μM SCH772984 (+) for 1 h, followed by addition of vehicle (−) or 100 nM PMA (+) for 2 h, and samples were subjected to immunoblot analysis for the expression/phosphorylation of the indicated proteins. Note that SCH772984 is an ATP-competitive inhibitor and does not prevent phosphorylation of ERK by MEK. B, IEC-18 cells were treated with 100 nM PMA for 6 h in the presence or absence of SCH772984 as indicated and DNA content/cell cycle distribution was determined by flow cytometric analysis. The percentage of cells in G₁, S, and G₂/M phase is shown below the DNA histograms. C, i, IEC-18 cells, cultured overnight in medium containing 0.5% FBS (serum-starved), were treated (10 min) with vehicle (−), 50 ng/ml EGF, or 100 nM PMA in the absence or presence of 4 μM Gö6976 as indicated, prior to Western blot analysis. The graph to the right of the blots shows densitometric analysis of relative levels of pERK normalized to loading control (±SD, n = 5, ∗p ≤ 0.05; ∗∗p ≤ 0.01); ii, IEC-18 cells were treated with PMA for 6 h in the absence or presence of 4 μM Gö6976 and analyzed for the expression of PKCα, δ, or ε by Western blotting. D, Serum-starved IEC-18 cells were pretreated for 1 h with 4 μM Gö6976 or 5 μM BIM I as indicated, before treatment with vehicle (−) or EGF for 10 min. E, serum-starved IEC-18 cells were treated with PMA or EGF for the indicated times and subjected to Western blot analysis for pERK and total ERK. All data are representative of at least three independent experiments. BIM I, bisindolylmaleimide I; PMA, phorbol 12-myristate 13-acetate.